1. Field of the Invention
Neuroblast tumor (hereinafter referred to simply as NB) is a malignant tumor specific to infancy and curable only when discovered at an early stage. Therefore, early discovery and diagnosis are desired. In the case of true disease, vanillylmandelic acid (hereinafter referred to simply as VMA) and homovanillic acid (hereinafter referred to simply as HVA) as metabolic products of catechol amine are discharged into urine in substantial amounts. Accordingly, it is possible to diagnose NB in a high probability by measuring these two components in urine. In Japan, NB mass screening for children has been started since January 1985 at a governmental level.
The present invention relates to a method for simultaneously analyzing VMA, HVA and creatinine (hereinafter referred to simply as CRN) in a test solution quickly and accurately with high sensitivity for such NB mass screening, and to an apparatus useful for such a method.
2. Discussion of Background
In recent years, it has been common to use fast liquid chromatography for the measurement of VMA and HVA. In this method, the separation is conducted by reversed phase chromatography or anion exchange chromatography and the detection is conducted by a ultraviolet absorption measuring method (A. Yoshida, et al, J. Chromatogr., 227,162 (1982)), an electrochemical measuring method (M. H. Joseph, et al, J. Chromatogr., 226,361 (1981)) or a fluorescence measuring method (T. G. Rosano, et al, Clin. Chem. 27,228 (1981)).
However, in the test solution, there exist acidic components such as uric acid and other organic acids and basic components such as polyamines, in addition to the object components. These inclusions are likely to interfere with the determination. Accordingly, it used to be difficult to directly inject the test sample, and it used to be required to have some pretreatment conducted. For such pretreatment, it has been common to employ a method wherein urine or urine-absorbed filter paper is extracted with ethyl acetate or with a buffer solution. However, each of such methods involves manual operation and thus has drawbacks such that not only the operation is cumbersome but also the recovery rates are likely to be irregular, and it is not possible to completely eliminate the influence of inclusions.
On the other hand, in the determination of the quantity of a certain component in urine, it is common to adopt a creatinine ratio (CRN ratio) as a means for correction against the change of the amount of urine. Likewise, in the quantitative determination of VMA and HVA, it is necessary to preliminarily measure the CRN concentration in urine. In the conventional methods, the measurement of CRN was conducted by a method entirely independent from the system for the measurement of VMA and HVA, whereby the analysis of data was not only complex but also time-consuming. Therefore, the simplification has been desired.
As mentioned above, the conventional methods require a cumbersome pretreatment operation such as solvent extraction of VMA and HVA. Further, since the CRN analysis used to be impossible in a single series of measuring system, it took a long period of time for the overall analysis.